Electronic Journal of Biotechnology ISSN: 0717-3458

2010 by Pontificia Universidad Catlica de Valparaso -- Chile

Vol.13 No.2, Issue of March 15, 2010

Received May 11, 2009 / Accepted October 14, 2009

RESEARCH ARTICLE

DOI: 10.2225/vol13-issue2-fulltext-1

Simultaneous saccharification and fermentation process of different

cellulosic substrates using a recombinant Saccharomyces cerevisiae
harbouring the -glucosidase gene
Vernica Ferreira
Laboratorios de Desenvolvimento de Bioprocessos
Departamento de Engenharia Bioqumica
Universidade Federal do Rio de Janeiro
Rio de Janeiro, Brasil

Mariana de Oliveira Faber

Laboratorios de Desenvolvimento de Bioprocessos
Departamento de Engenharia Bioqumica
Universidade Federal do Rio de Janeiro
Rio de Janeiro, Brasil

Sabrina da Silva Mesquita

Laboratorios de Desenvolvimento de Bioprocessos
Departamento de Engenharia Bioqumica
Universidade Federal do Rio de Janeiro
Rio de Janeiro, Brasil

Nei Pereira Jr.*

Laboratorios de Desenvolvimento de Bioprocessos
Departamento de Engenharia Bioqumica
Universidade Federal do Rio de Janeiro
Rio de Janeiro, Brasil
E-mail: nei@eq.ufrj.br
Financial support: Brazilian Petroleum Company (PETROBRAS); The Brazilian Council for Research (CNPq) and the Rio de Janeiro Foundation for
Science and Technology (FAPERJ).
Keywords: bioethanol, lignocellulosic biomass, sugarcane bagasse derived cellulignin.
Abbreviations: AVICEL: crystalline cellulose
CMC: carboxymethyl cellulose
FPU: filter paper activity
SCBPCL: sugar cane bagasse pre-treated cellulignin
SSF: saccharification and fermentation process

In Brazil, the production of ethanol from sugarcane

produces large amounts of lignocellulosic residues
(bagasse and straw), which have been driving research
and development for the production of second
generation ethanol. In the present work, a recombinant
Saccharomyces cerevisiae strain expressing the glucosidase gene from Humicola grisea was used for
ethanol production from three different cellulosic
sources by simultaneous saccharification and
fermentation. Initially, a enzymatic pre-hydrolysis step
was done with a solid:liquid ratio of 1:4, and an
enzymatic load of 25 filter paper activity (FPU).g-1 of
cellulosic substrate. Using sugarcane bagasse pretreated
cellulignin, crystalline cellulose and carboxymethyl
cellulose, 51.7 g L-1, 41.7 g L-1 and 13.8 g L-1 of ethanol
was obtained, respectively, at the end of 55 hrs of

fermentation. The highest ethanol productivity (0.94 g

L-1 hrs-1) was achieved using sugarcane bagasse
pretreated cellulignin. The use of a recombinant S.
cerevisiae led to extremely low glucose concentrations
when compared to other works reported in literature.
The search for new technologies for converting plant
biomass into alternative biofuels is leveraged by many
social and environmental problems associated with the use
of fossil fuels and their exploration. Bioethanol is one the
most promising biofuels from renewable resources.
Fermentation-derived ethanol can be produced from sugar,
starch or lignocellulosic biomass. Sugar and starch based
feedstocks are currently predominant at the industrial level
and they are so far economically favorable (Krishna et al.
2001; Shen et al. 2008; Sukumaran et al. 2009).

*Corresponding author

This paper is available on line at http://www.ejbiotechnology.info/content/vol13/issue2/full/1/

Ferreira, V. et al.

In Brazil, bioethanol is produced by the sucrose

fermentation from sugarcane. Great amounts of sugarcane
bagasse are produced in this process. In the 2007/2008
harvest season, 496 Millions of metric tons of sugarcane
were processed, producing 22 Million cubic meters of
bioethanol (MAPA - www.agricultura.gov.br). The
sugarcane bagasse has a complex structure, composed of
40-50% cellulose, 25% hemicellulose and 25% of lignin. Its
conversion into fermentable sugars is possible using
thermal pre-treatment, followed by chemical or enzymatic
hydrolysis (Hernndez-Salas et al. 2009). One of the routes
for second generation ethanol production from sugar cane
bagasse starts with a thermal pre-treatment, which aims at
disorganizing and fractioning the lignocellulosic complex.
This pre-treatment can be integrated with an acid catalyzed
step in mild conditions as for hydrolyzing the hemicellulose
component, generating a pentose-rich liquid phase, which
can be fermented by an adequate microorganism (e.g.,
Pichia stipitis). The remaining solid material is composed
mainly of cellulose and lignin, named cellulignin. This
solid residue undergoes an alkaline delignification process
to ensure the increase of the cellulose saccharification
efficiency. Cellulose saccharification can be carried out
either separately (SHF process) or simultaneously (SSF
process) to the hexose fermentation step (Chen et al. 2007;
Olofsson et al. 2008).

glucose (Sukumaran et al. 2009). The Simultaneous

Saccharification and Fermentation processes, firstly
described by Takagi et al. (1977), combines enzymatic
hydrolysis of cellulose with simultaneous fermentation of
its main derived sugar (glucose) to ethanol. In this process,
the stages are virtually the same as in the separate
hydrolysis and fermentation system, except that both are
performed in the same bioreactor. Thus, the presence of
yeast together with the cellulolytic enzyme complex
reduces the accumulation of the inhibiting sugars within the
reactor, thereby increasing the yield and the
saccharification rates. Another advantage of this approach
is that a single bioreactor is used for the entire process,
therefore reducing the investment costs. In addition, the
presence of ethanol causes the medium to be less
vulnerable to invasion by undesired microorganisms
(Ballesteros et al. 2004; Olofsson et al. 2008).

Cellulose, the major component of plant biomass, is a

polysaccharide enzymatically hydrolysable by three
different
cellulolytic
enzymes:
endoglucanases,
exoglucanases and -glucosidases (Vsquez et al. 2007). It
is well documented that this enzymatic complex system is
inhibited by its final hydrolysis products, particularly by

Microorganism

Figure 1. Production of
Humicola grisea.

The present study aimed at evaluating the simultaneous

saccharification and fermentation of three different
cellulosic sources (carboxymethyl cellulose, crystalline
cellulose and sugar cane bagasse pre-treated cellulignin)
using a recombinant Saccharomyces cerevisiae harbouring
the -glucosidase gene from Humicola grisea.
MATERIALS AND METHODS

A strain of Saccharomyces cerevisiae containing the glucosidase gene from Humicola grisea was obtained from
the Molecular Biology Group of Brasilia University
(Brazil) and was kindly provided to be use in this work.

-glucosidase by a recombinant strain of Saccharomyces cerevisiae harbouring bgl4 gene from

SSF process for ethanol production using S. cerevisiae harbouring the -glucosidase gene

Table 1. Composition of sugar

pretreated cellulignin (SCBPCL).

cane bagasse

concentration in the medium was fixed at 30% saturation,

through agitation in the range of 200-550 rpm and air flow
rate in the range of 0.5-1.0 L min-1.
Substrates

Composition

The cellulosic sources evaluated were: carboxymethyl

cellulose (Sigma), crystalline cellulose (Reagen) and sugar
cane bagasse pre-treated cellulignin, which was obtained
after diluted acid pre-treatment of sugar cane bagasse
(H2SO4, 1%v/v; solid:liquid ratio, 1 g:2 mL; temperature,
121C; and time of 40 min), followed by alkaline
delignification (NaOH, 4% w/v; solid:liquid ratio, 1 g:20
mL; temperature, 121C; and time, 30 min) of the
remaining solids, as reported by Vsquez et al. (2007). The
contents of sugars, lignin and ash were determined
according to the National Renewable Energy Laboratory
(Sluiter et al. 2004) and Ververis et al. (2007).

% of dry matter SD

Glucan

67.968 1.309

Hemicellulose

12.197 0.945

Lignin

9.303 0.617

Ash

3.501 0.423

SD: standard deviation.

The bgl4 gene from Humicola grisea was cloned in the

pGEM-T Easy (Promega) plasmid and the pYBG4 plasmid
was used to transform the Saccharomyces cerevisiae yeast.
Growth conditions
Active cultures for inoculation were prepared by growing
the organism on a rotary shaker at 200 rpm for 18 hrs at
30C, in a growth medium containing: yeast nitrogen base
(Difco), 0.34 g L-1; aspartic/glutamic (Vetec), buffer 0.4 g
L-1 and glucose (Vetec), 20 g L-1.
Enzymatic production
The enzymatic production was performed in an
instrumented 2 L bioreactor model Biostat (B. Braun
Biotech International), with control of temperature, pH,
dissolved oxygen level and foam. After the yeast growth,
the cell suspension was centrifuged and resuspended in 1 L
of medium for enzymatic production (yeast extract (Difco),
5.64 g L-1; aspartic/glutamic (Vetec) buffer, 0.4 g L-1 and
glucose (Vetec), 20 g L-1). The medium temperature was
maintained at 37C and pH adjusted to 6.0 with automatic
addition of a 2M NaOH solution. The dissolved oxygen

Cellulase source
Cellulase complex derived from Trichoderma reesei
(MULTIFECT) was provided by Genencor (Danisco
Division).
Simultaneous saccharification and fermentation
assays
The SSF experiments were carried out batchwise in 250 mL
conical flasks and in a 1 L bioreactor (Bioflo III, New
Brunswick Scientific Co.). The conical flasks and the
bioreactor were added with 10 g and 100 g dry cellulosic
source, and 40 mL and 400 mL of sodium citrate buffer pH
5.0, respectively, and the media sterilized at 121C/20 min.
A cellulase load, corresponding to 25 FPU.g-1 dry cellulosic
source,was transferred to the systems, prior to the SSF
process as for an enzymatic hydrolysis pre-treatment, which
was performed at 47C for 12 hrs. Thereafter, the
temperature was reduced to 37C and a yeast cell
suspension was added into the fermentation systems in a
concentration of 2 g L-1.

Table 2. Concentrations of glucose and ethanol considering complete cellulose hydrolysis and fermentation efficiency.

Cellulose Content
(%)

Corresponding Glucose
Concentration (g L-1)

Theoretical ethanol concentration

(g L-1)

CMC

100

220

112.42

SCBPCL

68

149.6

76.45

AVICEL

100

220

112.42

Cellulosic substrate

CMC: carboxymethyl cellulose; SCBPCL: sugar cane bagasse pre-treated cellilignin; AVICEL: crystalline cellulose.

Ferreira, V. et al.

The composition of sugar cane bagasse pre-treated

cellulignin is present in Table 1. These values were used in
the calculation of the sugar recovery and ethanol yield.
Considering
complete
cellulose
hydrolysis
and
fermentation efficiencies, the theoretical glucose and
ethanol concentrations obtained in the spent media are
displayed in Table 2. These values are of importance as for
estimating the practical fermentation efficiency.
Enzymatic production

Figure 2. Simultaneous saccharification and fermentation of

different
cellulosic
sources
by
a
recombinant
Saccharomyces cerevisiae harbouring Humicola grisea bgl4
gene for second generation ethanol production in shaken
flasks. PH: prehydrolysis; CMC: carboxymethycellulose;
AVICEL: crystalline cellulose and SCBPCL: sugar cane bagasse
pre-treated cellulignin.

Cell disruption
Recombinant cells were subjected to successive washes
with sodium acetate buffer pH 6.0 followed by freezing and
subsequent shearing with glass beads by vortex. The
intracellular extract was stored at 4C for subsequent
quantification of the intracellular -glucosidase activity.
Analytical method
Filter paper activity (FPU) was measured as described by
Ghose (1987). One International Filter Paper Unit of
enzyme activity was defined as the amount of enzyme
required for producing one micromol of glucose per
minute. For the determination of -glucosidase activity in
cellobiose, 50 L of enzymatic extract (intra or
extracellular), previously diluted, were incubated with 50
L in a 15 mM cellobiose solution, pH 6.0 for 30 min at
40C in a thermostatized bath (Dubnoff). After the reaction
time, the enzyme deactivation (100C for 10 min) was done
followed by the quantification of glucose concentration
using the enzymatic-colorimetric assay (GOD-POD). The
cell concentration was determined using the correlation
between dry cell concentration and absorbance at 600 nm.
Cellobiose, glucose and ethanol were analyzed with HPLC
Waters chromatograph equipped with a refractive index
detector using a Bio-Rad aminex HPX-87H column at 60C
and water as mobile phase at a flow rate of 0.4 mL min-1.

The recombinant -glucosidase production in bioreactor

was carried out batchwise in aerobic conditions, with the
objective to analyze the cell growth and the enzymatic
production rates (Figure 1). The specific cell growth rate
between 3 and 9 hrs was 0.116 hrs-1. With an average
consumption rate of 2.27 g L-1 hrs-1, after 9 hrs of
cultivation, glucose was nearly depleted, and the specific
growth rate was reduced significantly. The recombinant
strain expresses -glucosidase constitutively, producing
mainly intracellular activity, and in a less extent
extracellular activity. The total enzymatic production
(intracellular and extracellular) was roughly 1100 U L-1 at
the end of 24 hrs of cultivation, corresponding to an
enzymatic yield on grown cells (YP/X) of 170 U g-1. First,
the recombinant strain expressed the enzyme synthesis
(intra and extracellular) concurrently with cell growth and
ethanol production (fermentative phase). The intracellular
activity continued to be expressed even after total glucose
depletion, when the cell consumed ethanol as carbon source
(aerobic phase). On the other hand, extracellular activity
was observed up to 10 hrs of cultivation, remaining
constant thereafter.
The results point out this strain of Saccharomyces
cerevisiae, expressing the -glucosidase gene, with great
potential for ethanol production by the simultaneous
saccharification and fermentation process (SSF), since this
particular strain is capable of supplying the medium with glucosidase, enriching the commercial enzymatic blends
used in the SSF process.
Chen et al. (2007) and Hodge et al. (2008) report the need
to supplement commercial cellulases used in SSF process
with -glucosidase. Vsquez et al. (2007) also observed the
accumulation of cellobiose nearly to 10 g L-1 during the 10
first hours of fermentation, when using the bakery S.
cerevisiae strain for bioethanol production in SSF process
with sugarcane bagasse pre-treated cellulign, and had to
add additional -glucosidase in their experiments to reduce
the cellobiose concentration.
Simultaneous saccharification and fermentation
assays with the recombinant yeast

RESULTS AND DISCUSSION

Experiments in conical flasks. The SSF process was

Glucan contents in the cellulosic sources

preceded by 12 hrs-enzymatic prehydrolysis, after which

the concentrations of glucose and cellobiose generated from
4

SSF process for ethanol production using S. cerevisiae harbouring the -glucosidase gene

Table 3. Concentration of sugars after enzymatic prehydrolysis of different cellulosic substrates. Solid:liquid ratio, 1:4;
temperature, 47C; agitation speed: 200 rpm and time, 12 hrs.

Concentration SD (g L-1)
Cellulosic Substrate
Cellobiose

Glucose

0.00

25.10 0.45

SCBPCL

1.28 0.62

69.79 2.83

AVICEL

8.62 0.24

59.54 0.21

CMC

SD: standard deviation; CMC: carboxymethyl cellulose; SCBPCL: sugar cane bagasse pre-treated cellilignin; AVICEL: crystalline cellulose.

different cellulosic substrates were determined (Table 3).

While carboxymethyl cellulose (CMC) prehydrolysis
produces exclusively glucose, sugar cane bagasse pretreated cellulignin (SCBPCL) and crystalline cellulose
(AVICEL) prehydrolyses generate both glucose and
cellobiose. It is likely that the amount of glucose produced
using SCBPCL and AVICEL led to the inhibition of glucosidase, leaving a residual cellobiose concentration.
According to hgren et al. (2007), -glucosidases are
strongly inhibited by glucose, while endo- and exoglucanase are inhibited by cellobiose. The difference
between the produced sugar concentrations with SCBPCL
and AVICEL in comparison with CMC in the prehydrolysis
step is due to the type of cellulose and the enzymatic
activities present in the commercial pool, being also
strongly dependent on the sort of lignocellulose pretreatment (Hodge et al. 2008). Sukumaran et al. (2009) also
obtained different fermentable sugar concentrations at the

Figure 3. Simultaneous saccharification and fermentation of

sugarcane bagasse pre-treated cellulignin by a recombinant
Saccharomyces cerevisiae harbouring Humicola grisea bgl4
gene for second generation ethanol production in
bioreactor. PH: enzymatic prehydrolysis.

end of the prehydrolysis process using water hyacinth

biomass, rice straw and sugar cane bagasse, resulting in
14.2 g L-1, 26.3 g L-1 and 17.8 g L-1, respectively, indicating
the close relationship between enzymatic pools and
cellulosic sources.
After the prehydrolysis each cellulosic substrate was
inoculated with the recombinant Saccharomyces cerevisiae
and the ethanol production was monitored for 65 hrs of SSF
process (Figure 2). As it can be expected SCBPCL and
AVICEL were the best substrates for SSF process, resulting
in higher values of ethanol concentration when compared
with CMC, since the former cellulosic sources resulted in
higher available fermentable sugars than the latter source in
the prehydrolysis step (Table 3). Cellobiose, glucose,
ethanol concentrations and fermentation efficiencies, at the
end of the SSF process, for the three investigated cellulosic
sources are shown in Table 4. In all of them, celobiose was
totally converted in glucose, and the values of volumetric
productivity were 1.18 g L-1 hrs-1, 0.95 g L-1 hrs-1 and 0.33
g L-1 hrs-1 for SCBPCL, AVICEL and CMC, respectively,
at the 43th hrs after cell inoculation. The presence of
glucose in the medium at the end of the SSF process
indicates the continuation of the catalytic activity of the
celullase complex (Ballesteros et al. 2004), as it was
noticed in the experiments herein done (Table 4).
Bench bioreactor experiments. Since SCBPCL provided
the best results in shaken flasks for both enzymatic
prehydrolysis and SSF process, this cellulosic source was
selected for experiments with the recombinant yeast to
evaluate its performance in bioreactor, and quantify more
accurately the process variables. At the end of the
enzymatic prehydrolysis, the medium contained 95.5 g L-1
of glucose and 29.5 g L-1 of cellobiose. The bioreactor was
then inoculated with 2 g L-1 of the recombinant S.
cerevisiae (Figure 3), starting the SSF process. After 78 hrs,
an ethanol concentration of 60 g L-1 was achieved and no
accumulation of cellobiose was observed, demonstrating
the effective action of the -glucosidase expressed by the
recombinant strain.

Ferreira, V. et al.

Table 4. Residual concentration of sugars and maximum ethanol production in SSF process.

Final concentration (g L-1)

Cellulosic substrate

FE (%)
Cellobiose

Glucose

Ethanol

CMC

0.00

0.59

15.15

13.5

SCBPCL

0.00

0.92

52.69

68.9

AVICEL

0.00

1.15

42.45

37.8

FE: fermentation efficiency (obtained ethanol/theoretical ethanol); CMC: carboxymethyl cellulose; SCBPCL: sugar cane bagasse pre-treated
cellilignin; AVICEL: crystalline cellulose.

The ethanol production in bioreactor also validated the

results obtained using shaken flasks, resulting in similar
performance (average volumetric productivity of 1 g L-1
hrs-1 for both systems). The results obtained in the present
work are of interest, since they bring the solution for
reducing the residual cellobiose concentrations normally
found in SSF processes of lignocellulosic biomass. Besides
this, the ethanol concentrations and the ethanol efficiency
achieved are far greater than others reported in recent
literature. Hernndez-Salas et al. (2009) achieved 12.5 g L-1
of ethanol using the SSF process with sugarcane bagasse
previously acid-pre-treated and NaOH-delignified, after 48
hrs of process, corresponding an ethanol efficiency of
32.6%. Similar results were obtained by Sukumaran et al.
(2009), achieving at the end of SSF process an ethanol
concentration of 12.3 g L-1 using rice straw pre-treated with
alkali and enzymatically hydrolyzed, after 24 hrs of
process, which corresponded to an ethanol efficiency of
40.3%.

production but also for the ethanol fermentation by the SSF

process, which glucose is continuously generated in the
production medium, assuring the extracellular expression of
-glucosidase. The best results of ethanol production using
SSF were obtained with the most complex cellulosic source
(sugar cane bagasse pre-treated cellulignin), achieving 51.7
g L-1 and 60 g L-1, in shaken flasks and in bioreactor,
respectively. These results show the biotechnological
potential of the recombinant strain of S. cerevisiae for the
production of second generation ethanol.

CONCLUDING REMARKS

CHEN, H.Z.; XU, J. and LI, Z.H. Temperature cycling to

improve the ethanol production with solid state
simultaneous saccharification and fermentation. Applied
Biochemistry and Microbiology, February 2007, vol. 43,
no. 1, p. 57-60.

The major bottleneck in the second-generation bioethanol

production is the high cost of the cellulolytic enzymes.
Most commercially available enzymatic pools do not show
sufficient
-glucosidase activity for an efficient
simultaneous saccharification and fermentation process. In
this work, a recombinant S. cerevisiae strain harbouring the
blg4 gene from Humicola grisea was used. Apart from
producing ethanol in high levels, the recombinant strain can
complement the used commercial enzymatic pools with glucosidase, and thus guaranteeing the total conversion of
cellobiose to glucose during SSF process. The glucosidase production by submerged fermentation carried
out batchwise resulted in a global enzyme activity (intra
and extracellular) of 1100 U L-1. The intracellular enzyme
continued to be produced even after glucose depletion,
while the extracellular activity was detected only when
there was glucose in the medium. This fact resulted in a
boost in the use of this strain not only for enzymatic

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